The heterogeneity of the elongation factor EF1 from wheat embryos

Biochimica et Biophysica Acta, 335 (1974) 275-283

Elsevier ScientificPublishingCompany,Amsterdam - Printed in The Netherlands BBA 97892

THE HETEROGENEITY OF THE ELONGATION FACTOR EF1 FROM WHEAT EMBRYOS

G. A. LANZANI, R. BOLLINIand A. N. SOFFIENTINI Laboratorio Virus e Biosintesi Veyetali C.N.R., Milano (Italy)

(Received August 2nd, 1973)

SUMMARY The elongation factor EF1 from wheat embryo, can be separated in several forms with different molecular sizes. A heavy form was purified from other heterogeneous species with smaller molecular sizes. While the purified heavy form can join directly in a binary complex with GTP, it has to be converted into a light species for the formation of the ternary complex aminoacyl-tRNA-GTP-EF1.

INTRODUCTION The binding of aminoacyl-tRNA to ribosomes during the elongation phase of polypeptide synthesis is operated in Escherichia coli by the factors Tu and Ts. Their molecular weights have been found to be 40 000 and 19 000, respectively [1 ]. In eukaryotic systems the same functions are performed by the factor EF1. Some molecular characteristics of EF1 are known; the enzyme from reticulocytes has a molecular weight of 186 000, and can be dissociated into inactive subunits of a molecular weight of 62 000 [2]; from rat liver it has been reported to exist in multiple forms with molecular weights ranging from 100 000 to more than 300 000 [3, 4]; in calf brain there is evidence of two species of EF1 with different molecular weights [5]. In wheat embryos Allende [6] and Legocki [7] reported that EF1 is present in two forms of different molecular weight. Moreover, Allende [6] showed that GTP and GDP can transform the heavy form into the lighter form. We now report a new procedure for the purification of the heavy form of EF1 from wheat embryos. The data here presented show that the heavy species can form a binary complex with GTP, and that the heavy form has to be transformed into the light species for the formation of the ternary complex (aminoacyl-tRNA-GTP-EF1). METHODS AND MATERIALS Preparation o f the ribosomes

300 g of wheat embryos obtained by the method of Johnston and Stern [8] were homogenized with 500 ml of Buffer A (0.25 M sucrose, 50 mM Tris-HCl, 10 mM MgCI2, 25 mM KCI, 5 mM 2-mercaptoethanol, pH 7.5) in a Braun mixer

276

for 5 min, at low speed. The homogenate was centrifuged in a Sorvall refrigerated centrifuge for 30 rain at 5000 x g ; the supernatant was maintained for 4 h at 0 °C; another precipitate was formed and it was centrifuged away at 12 000 x g for 30 rain. The 12 0 0 0 x g supernatant was centrifuged in a Spinco L 2 ultracentrifuge for 3 h at 100 000 xg. The 100 000 x g supernatant was used for the purification of the elongation factors. The 100 000 x g pellet was suspended in 80 ml of Buffer B (Buffer A without sucrose). The ribosome suspension was centrifuged for 30 min at 12 000 x g and, from this clarified solution, the ribosomes were pelleted by centrifugation for 3 h at I00 000x9. The ribosome pellet was suspended in 8 ml of Buffer B. These ribosomes, not completely free from transfer factors, were further purified by DEAEcellulose chromatography as already described [9]. Only DEAE-chromatographed ribosomes, completely deprived of transfer factors, were used for the assay during the purification of EF1.

Purification of the elongation factor EF1 The 100 000x9 supernatant was passed through a Sephadex G-25 column, conditioned with 10 mM Tris-HC1 and 1 mM 2-mercaptoethanol. The protein peak, eluted in the void volume, was precipitated by adding 56.1 g of of (NH4)zSO 4 per 100 ml solution. After centrifugation at 12 0 0 0 x 9 for 20 rain, the pellet was resuspended three times with 100 ml (each time) of a solution containing 35 g of (NH4)2SO 4 in 100 ml of 10 mM Tris, 1 mM 2-mercaptoethanol buffer, pH 7.8. After centrifugation at 12 000 x 9, 20 min each time, the supernatants were discarded. The final pellet was resuspended three times with 100 ml (each time) of a solution containing 15 g of (NH4)2SO 4 in 100 ml of the buffer already described. After centrifugation the 15 ~ (NH4)2SO4-washed pellet was discarded and the three solutions were pooled and precipitated by adding 39 g of (NH4)2SO 4 per 100 ml solution. The precipitate was centrifuged and the pellet containing the elongation factors was dissolved in 50 ml of 20 mM Tris, 1 mM 2-mercaptoethanol, 8 mM MgC1 z, 200 mM KC1 buffer, pH 7.5 (Buffer C), and dialyzed overnight against Buffer C. 70 ml of this solution were gel filtrated on a Sephadex G-200 column (80 c m x 10 era) conditioned with Buffer C; the flow rate was maintained at 300 ml/h with an LKB pump. 20-ml fractions were collected and tested for the Phe-tRNA binding to ribosomes and for phenylalanine polymerization activities. The tubes containing only binding activity and no translocation activity were pooled, precipitated at 80 ~ (NH4)2SO 4 saturation and dissolved in 10 ml of a 50 mM potassium phosphate, I mM dithiothreitol buffer, pH 7.5 (Buffer D), and dialyzed overnight against Buffer D. 12 ml of this solution were passed through a hydroxylapatite column (30 c m x 1 cm), conditioned with Buffer D, and eluted with a linear gradient (formed in a Varigrad apparatus) with 150 ml of a Buffer D and 150 ml of 300 mM potassium phosphate, 1 mM dithiothreitol buffer, pH 7.5. A flow rate of 15 ml/h was maintained with an LKB pump. Fractions of 10 ml were collected. [1~C]Phe-t RNA preparation [14C]Phe-tRNA was prepared from partially purified tRNA Ph~ of wheat germ by the method of Vold and Sypherd [10]. The product contained 304 pmoles (24100 cpm) of [14C]Phe-tRNA per A26o ,m unit.

277

EF1 activity The EF1 activity was assayed by means of the enzymatic binding to ribosomes of [14C]Phe-tRNA by the method of Niremberg and Leder [11], at a 7.5 mM Mg 2÷ concentration, where the non-enzymatic binding to the ribosomes was negligible [9]. 0.1 ml reaction mixture contained: 50 mM Tris-HCl, pH 7.8, 7.5 mM MgC12, 25 mM KCI, 2 mM 2-mercaptoethanol, 1 mM ATP, 0.5 mM GTP, 5 mM phosphoenolpyruvate, 2 #g phosphoenolpyruvate kinase, poly(U) (0.02 pmole P), 0.2 .4260 n m unit of [~4C]Phe-tRNA (60.8 pmoles), 3 A26 o ,m units of ribosomes, and variable amounts of EF1. After incubation at 32 °C for 20 min the tests were added with 1 ml of diluting buffer (10 mM Tris-HC1, pH 7.8, 7.5 mM MgC12, 25 mM KC1) at 0 ° C, passed through nitrocellulose filters and washed three times with 2 ml of the diluting buffer. The filters were dried and counted in a Beckman CPM 100 scintillator in PPO-POPOP scintillation medium. One unit of EF1 is the amount of enzyme which binds one pmole of Phe-tRNA to ribosomes under the conditions described. EF2 activity The EF2 activity was determined by testing the ability to complement the EF1 factor in poly(U)-directed polyphenylalanine synthesis [12]. The reaction mixtures were the same as described for EF1 activity, except that 20 units of EF1 devoid of EF2 activity were always present, and different amounts of protein with EF2 activity were added. After incubation at 32 °C for 20 rain, 0.1 ml of l0 ~o trichloroacetic acid were added; the mixtures were maintained for 20 min at 90 °C; the hot trichloroacetic acid-insoluble material was collected and washed on nitrocellulose filters with 5 ~ trichloroacetic acid, dried and counted. Formation of EF1-GTP and Phe-tRNA-GTP-EF1 The incubation was performed according to Tarragb et al. [13], except that 4 . 8 . 1 0 - 4 M [3H]GTP (22 Ci/mole), 1330 pmoles of [14C]Phe-tRNA (40 Ci/mole) and 560 units of EF1 (purified heavy form) were used. The complexes were separated on a Sephadex G-150 column by the method of Moon et al. [5]. Polyacrylamide disc gel electrophoresis Electrophoresis of the enzyme was done in 7% polyacrylamide gels (pH 8.3) as described by Davis [14]. Ultracentrifugation Sedimentation velocity measurements were carried out in a Spinco model E analytical ultracentrifuge equipped with schlieren optics. The measurements were performed in Buffer B at 14 °C and at a speed of 60 000 rev./min in a standard 12ram double sector cell with sapphire windows. Protein concentration The protein concentration was determined according to the method of Lowry et al. [15] using crystalline bovine serum albumin as a standard. [14C]Phenylalanine and [3H]GTP were purchased from NEN; ATP, phosphoenolpyruvate, phosphoenolpyruvate kinase, ferritine, catalase and bovine serum albumin from Boehringer; poly(U) from Miles; Sephadex G-200, G-150 and G-25 from Pharmacia. The nitrocellulose filters were Millipore H.A.W.P., 0.45 pm pore diameter. The other chemicals were analytical grade from Merck.

278 RESULTS

Purification of EF1 Table I reports the results of the different steps of purification. The 100 000 × # supernatant was precipitated at 80 ~ (NH4)2SO 4 saturation. The pellet was at first suspended in a 35 ~o (NH4)2SO4 solution, no EF1 activity was solubilized; it was then suspended in a 15 % (NH4)2SO 4 solution, 92 ~ of the EF1 activity was solubilized. This solution contained both EF1 and EF2 activities as it could perform the poly(U)directed polyphenylalanine synthesis with ribosomes completely deprived of transfer factors. The EFI activity could be separated from the EF2 activity by gel filtration on a preparative Sephadex G-200 column (Fig. 1). The Fractions 11-25 (Peak I) contained EF1 activity and no EF2 activity, because they were able to catalyze the binding of [14C]Phe-tRNA to ribosomes but did not form polyphenylalartine. After this peak a shoulder of EF1 activity was present (Fractions 26-40). The Fractions 51-70 (Peak II) contained EF2 activity and no EFI activity since they did not TABLE I P U R I F I C A T I O N O F EFI F R O M W H E A T E M B R Y O S Fraction

100 000 × # supernatant 15 ~ (NH4)2SO4 extraction Sephadex G-200 gel filtration (Peak I) Hydroxylapatite (Peak II)

Volume (ml)

Total protein (rag)

540

4100

48 13 4

Specific activity (units/ m g protein)

Total units ( × 1000)

Yield (~)

8.2

33.6

100

1240

24.8

30.8

92

70 6

268.4 1870.0

18.8 11.2

56 33

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10

20

30

40

50

60

70

80

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100

1"10

FRACTION

Fig. 1. Preparative Sephadex G-200 gel filtration o f EF1. EF1 activity ( O - - - O ) , a n d E F 2 activity (V - - -V ) were determined as described in the text on 0.1-ml aliquots o f each fraction; -, absorbance at 280 n m .

279 show Phe-tRNA binding activity, but could complement EF1 for the polymerization of the phenylalanine. Immediately after the peak of EF2 activity, fractions showing EF1 activity were eluted (Fractions 81-100) (Peak III). These facts indicate that EF1 from wheat embryo cytoplasm is heterogeneous and the various forms have different molecular sizes. Fig. 2 shows the chromatography on a hydroxylapatite column of the Peak I obtained by gel filtration. The EF1 activity was resolved in two different peaks: the first was widely spread from Fractions 6-12, and the enzymatic activity of these fractions was very labile. The greater part of the original EF1 activity was eluted as a sharp peak centered in Fraction 16 (Peak II), and could be stored for months in small samples at --20 °C.

'0.15 o 50, J~ E ¢

-r

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o

a. 2 5 . o o E o.

-0.05

•, ~ . _ . _ d 5

-"~o / 1'0

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20

FRACTION

Fig. 2. Hydroxylapatite chromatography of EF1. EFI activity (0- - -0) was determined as described in the text on 0.l-ml aliquots of each fraction; , absorbance at 280 nm.

Purity and molecular size of the EF1 preparations Peak II from the hydroxylapatite chromatography was gel filtrated on an analytical Sephadex G-200 column calibrated with protein markers of different molecular weights. EF1 activity was eluted as a peak (Fig. 4A), with an etution volume between those of ferritine (mol. wt 540 000) and catalase (mol. wt 240 000). Certainly no EF1 activity corresponding to proteins with molecular weights lighter than catalase are present. Sedimentation velocity measurements in a Spinco E analytical ultracentrifuge of this preparation of EF! showed the presence of one peak with an S value of 8.7 at a concentration of 1.5 mg/ml (Fig. 5). From these data we can conclude that the EF1 preparation from Peak II of the hydroxylapatite column contains a heavy form of the enzyme, and that this form is homogeneous as far as the molecular size is concerned. Moreover the refiltration on Sephadex G-200 of the heavy form of EF1 demonstrates that this form cannot spontaneously change into lighter forms. Fig. 4B shows the behaviour, on the same column of Sephadex G-200, of Peak III from the preparative Sephadex G-200 gel filtration; this peak contains enzyme forms of smaller molecular weight than catalase; one of them is eluted at an elution volume similar to that of bovine serum albumin (tool. wt 67 000). Analytical disc electrophoresis of the two types of EF1 preparations is shown in Fig. 3. Peak II from the hydroxylapatite chromatography gave rise to two bands.

280

2

3

A

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1

2

3

1

1

1

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A

A 20

25

30

35

FRACTION

Fig. 3. Disc gel electrophoresis of two types of EF1 preparations. Gels were run at 2 mA per tube at 15 °C until bromophenol blue ran from the top to the bottom of the gel. Gels were stained with Coomassie blue in 1 ~o trichloroacetic acid solution (1 g/100 ml) and destained with 10 ~'/otrichloroacetic acid. (A) Peak II from the hydroxylapatite chromatography; (B) Peak Iii of the Sepbadex G-200 preparative column. 100 !~g of protein were used. Fig. 4. Analytical Sephadex G-200 gel filtration of heavy and light forms of EFI. The column (1.5 cm × 15 cm) was conditioned with 50 mM Tris-HC1, 60 m M NH~CI, 10 m M MgC12, 1 mM dithiothreitol, pH 7.5, and eluted with the same buffer. Fractions of 0.5 ml were collected. In (A) 0.2 ml (560 EF1 units) of Peak II of the hydroxylapatite chromatography were gel filtrated. In B 0.4 ml (330 EF1 units) of Peak III of the preparative Sephadex G-200 column were used. EFl ( O - O ) was determined on 0.1-ml aliquots of each fraction. The elution of ferritine (Arrow 1), catalase (Arrow 2) and bovine serum albumin (Arrow 3) were determined in separate runs. These two bands did not correspond to any of the bands obtained from the lighter f o r m s o f E F I c o n t a i n e d in t h e P e a k I I I o f t h e S e p h a d e x G - 2 0 0 p r e p a r a t i v e c o l u m n (Fig. 3B). T h i s is a n o t h e r i n d i c a t i o n t h a t i n P e a k I I f r o m t h e h y d r o x y l a p a t i t e c o lumn the lighter forms of EF1 were not present.

281

!

Fig. 5. Sedimentation velocity pattern of EFI. The purified heavy form of EF1 (Peak II of the hydroxylapatite chromatography) was scdimented at a concentration of 1.5 mg/ml in Buffer B as described in the text. Pictures were taken at 5 (A) and 21 min (B) after reaching the full velocity, with bar angles of 45 (A) and 35 degrees (B).

Formation of the ternary complex Phe-tRNA-GTP-EF1 We incubated the EF1 preparation (Peak II from the hydroxylapatite column) with [3H]GTP and [14C]Phe-tRNA in the conditions already described by Tarrag6 et al. [13] for the formation of the ternary complex. After the incubation the mixture was gel filtrated on a Sephadex G-150 column, calibrated with ferritine, catalase and bovine serum albumin (Fig. 6). The EF1 activity (determined as poly(U)-directed I

2

3

/;

!

Z6O

"180

a. w

'120 a. o

w

~ 2o

-60

o

E D.

20

30

40

50

60

70

80

FRACTION

Fig. 6. Sephadex G-150 gel filtration of EF1 after incubation with GTP and Phe-tRNA. The column (1.5 c m × 15 cm) was conditioned with 50 mM Tris-HCl, 60 mM NH~CI, 10 mM MgC12, 1 mM dithiothreitol, pH 7.5, and eluted with the same buffer. Fractions of 0.5 ml were collected, The column was charged with 1 ml o f the complex, formed as described in Materials and Methods. 0.1-ml aliquots of each fraction were counted in 10 ml of Bray's scintillation mixture, for aH and 14C. 71- - -El, [SH]GTP pmoles per fraction; • - - - • , [14C]Phe-tRNA pmoles per fraction; 0 - 0 , EF1 units per fraction, determined as described in Materials and Methods. The elution volume of ferritine (Arrow 1), catalase (Arrow 2), and bovine serum albumin (Arrow 3) were determined in separated runs.

282

Phe-tRNA binding to the ribosomes), the 3H and 14C content of each fraction was measured. The EF1 activity was present in two peaks. The EF1 activity of the first peak (maximum at Fraction 29) is eluted between ferritine and catalase, and shows an elution volume similar to the original form of the enzyme incubated. The other peak of EF1 activity has a maximum at Fraction 46 and has an elution volume similar to that of bovine serum albumin. 3H is present in the fractions corresponding to the first peak of EF1 activity, but no 14C is present in these fractions; therefore a ternary complex, containing the heavy form of the enzyme, was not observed. The fractions containing the second peak of EF1 activity, contain both 3H and 14C; the molar ratio 3H/~4C is very close to 1 in the first part of the peak (shaded area in Fig. 6), while in the second part of the peak the ratio diminishes because of the overlapping of free [~4C]Phe-tRNA. The appearance of EF1 activity with an elution volume corresponding to that of bovine serum albumin means that a portion of the heavy form of the enzyme was transformed into a lighter form during the incubation for the formation of the ternary complex. The contemporary presence of this light form of EF1 activity, of [3H]GTP and [14C]Phe-tRNA in equimolar quantities in the same zone of elution (shaded area of Fig. 6) is an indication of the formation of the complex Phe-tRNA-GTP-EFI. DISCUSSION

Previous works [6, 7] demonstrated that two forms of EF1 with different molecular sizes are present in wheat embryo cytoplasm. We succeeded in purifying a heavy form more than 200 times. This form, which appeared homogeneous by gel filtration, gave rise to two bands by analytical disc electrophoresis. The two bands, however, did not correspond to any of the bands obtained with the light form of EF1; either the gel filtration or the disc electrophoresis data exclude the presence of light EF1 forms in the heavy form preparation. The heavy species of EFI cannot form a ternary complex with GTP and PhetRNA unless it is transformed into a lighter form. We cannot exclude the possibility that an unstable ternary complex with the heavy species is at first formed; on the other hand the purified light species by itself can form a binary complex with GTP, but is unable to form a ternary complex (unpublished results). Therefore, the transformation of the heavy form into the lighter one is necessary to obtain stable ternary complex. Manzocchi et al. [16] found that the heavy form of EFI can be transformed into a lighter one in the presence of either GTP or GDP. The ternary complex formation with wheat embryo EF1 has been demonstrated by Tarragb et al. [13] using the gel filtration method, but they did not show which of the two forms of EF1 was present in the complex. Our failure to demonstrate ternary complex formation with the heavy form of wheat embryo EF1 by means of Sephadex G-150 gel filtration is in agreement with what has been found with the high molecular size EF1 from rabbit reticulocyte [17] and from rat liver [4]. The transformation of the heavy type of EF1 into a lighter form during the formation of the ternary complex is very similar to what has been found for calf brain EF1 [5].

283 REFERENCES 1 Haenni, S. L. (1972) in The Mechanism of Protein Synthesis and its Regulation, (Bosch, L., ed.), pp. 33-54, North Holland, Amsterdam 2 McKe,ehan, W. L. and Hardesty, B. (1969) J. Biol. Chem. 244, 4330-4339 3 Schneir, M. and Moldave, K. (1968) Biochim. Biophys. Acta 166, 58-67 4 Collins, J. F., Moon, H. M. and Maxwell, E. (1972) Biochemistry 11, 4187-4194 5 Moon, H. M., Redfield, B. and Weissbach, H. (1969) Proc. Natl. Acad. Sci. U.S. 69, 1249-1252 6 Allende, J . E . (1972) I.U.B./I.U.B.S., Joint Symposium on Nitrogen Metabolism in Plants (Campbell, P. N., ed.), July 10th-14th, Leeds, Biochemical Society Symposia, London 7 Legocki, A. B. 0972) I.U.B./I.U.B.S., Joint Symposium on Nitrogen Metabolism in Plants (Campbell, P. N., ed.), July 10th-14th, Leeds, Biochemical Society Symposia, London 8 Johnston, F. B. and Stern, J. (1957) Nature 175, 160-161 9 Lanzani, G. A. and Soffientini, A. (1973) Plant Sci. Lett. 1, 89-93 l0 Vold, B. S. and Sypherd, P. S. (1968) Plant Physiol. 43, 1221-1226 11 Niremberg, M. W. and Leder, P. (1964) Science 145, 1399-1407 12 Leder, P. (1971) in Methods in Enzymology, Vol. 20, pp. 302-306, Academic Press, New York 13 Tarragb, A., Monasterio, O. and Allende, J. E. (1970) Biochem. Biophys. Res. Commun. 41, 765-773 14 Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121,404-427 15 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265275 16 Manzocchi, L. A., Tarragb, A. and Allende, J. E. (1973) FEBS Lett. 29, 309-312 17 Ravel, J. M., Dawkins, R. C., Lax, S., Odom, O. W. and Hardesty, B. (1973) Arch. Biochem. Biophys. 155, 332-341